Transcript of Population attributable risk of aflatoxin-related liver ...
Population attributable risk of aflatoxin-related liver cancer:
Systematic review and meta-analysisEuropean Journal of Cancer
(2012) 48, 2125– 2136
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Population attributable risk of aflatoxin-related liver cancer:
Systematic review and meta-analysis q
Yan Liu a, Chung-Chou H. Chang b,c, Gary M. Marsh c, Felicia Wu
a,⇑
a University of Pittsburgh, Graduate School of Public Health,
Department of Environmental and Occupational Health, Pittsburgh,
PA, USA b University of Pittsburgh, School of Medicine, Department
of Medicine, Pittsburgh, PA, USA c University of Pittsburgh,
Graduate School of Public Health, Department of Biostatistics,
Pittsburgh, PA, USA
Available online 8 March 2012
09
do
q
(5 ⇑
59-8049/$ - see front matter
Corresponding author: Addr ccupational Health, Graduate ttsburgh,
100 Technology l.: +1 412 624 1306; fax: +1 E-mail address:
few8@pitt.e
2012 E
Drive, P 412 624 du (F. W
Abstract Background: Over 4 billion people worldwide are exposed to
dietary aflatoxins, which cause liver cancer (hepatocellular
carcinoma, HCC) in humans. However, the popula- tion attributable
risk (PAR) of aflatoxin-related HCC remains unclear. Methods: In
our systematic review and meta-analysis of epidemiological studies,
summary odds ratios (ORs) of aflatoxin-related HCC with 95%
confidence intervals were calculated in HBV+ and HBV individuals,
as well as the general population. We calculated the PAR of
aflatoxin-related HCC for each study as well as the combined
studies, accounting for HBV status. Results: Seventeen studies with
1680 HCC cases and 3052 controls were identified from 479 articles.
All eligible studies were conducted in China, Taiwan, or
sub-Saharan Africa. The PAR of aflatoxin-related HCC was estimated
at 17% (14–19%) overall, and higher in HBV+ (21%) than HBV (8.8%)
populations. If the one study that contributed most to het-
erogeneity in the analysis is excluded, the summarised OR of HCC
with 95% CI is 73.0 (36.0– 148.3) from the combined effects of
aflatoxin and HBV, 11.3 (6.75–18.9) from HBV only and 6.37
(3.74–10.86) from aflatoxin only. The PAR of aflatoxin-related HCC
increases to 23% (21–24%). The PAR has decreased over time in
certain Taiwanese and Chinese populations. Conclusions: In high
exposure areas, aflatoxin multiplicatively interacts with HBV to
induce HCC; reducing aflatoxin exposure to non-detectable levels
could reduce HCC cases in high- risk areas by about 23%. The
decreasing PAR of aflatoxin-related HCC reflects the benefits of
public health interventions to reduce aflatoxin and HBV. 2012
Elsevier Ltd. All rights reserved.
lsevier Ltd. All rights reserved.
National Cancer Institute National Institutes of Health. rtment of
Environmental and f Public Health, University of ittsburgh, PA
15219, USA. 3040. u).
1. Introduction
Aflatoxins are toxic and carcinogenic chemicals produced primarily
by the fungi Aspergillus flavus and A. parasiticus, which infect
food crops such as maize,
2126 Y. Liu et al. / European Journal of Cancer 48 (2012)
2125–2136
peanuts, and tree nuts. About 4.5 billion people world- wide are
exposed to dietary aflatoxins.1 Exposures are highest in tropical
and subtropical regions of the world, where maize and peanuts are
dietary staples and food storage conditions are
suboptimal.1,2
Aflatoxins are amongst the most potent naturally occurring human
hepatocarcinogens known. The Inter- national Agency for Research on
Cancer (IARC) has classified “naturally occurring mixes of
aflatoxins” as a Group 1 human carcinogen.3 Abundant
epidemiological evidence suggests that aflatoxin exposure
synergises with chronic hepatitis B virus (HBV) infection to
increase liver cancer (hepatocellular carcinoma, HCC) risk in
populations with both risk factors.4–8 More recently, toxicological
models for the mechanism of the synergism of these two risk factors
have emerged,9–11 and are sum- marised in Wild and Gong.12
Unfortunately, both high aflatoxin exposure and HBV are prevalent
in many parts of the developing world, particularly Asia and
Africa.
Previously, by compiling food consumption and afla- toxin
contamination data in multiple countries and conducting a
quantitative cancer risk assessment, we esti- mated that
25,200–155,000 (5–28%) annual HCC cases worldwide could be
attributed to aflatoxin exposure.13
This large range highlights the limitations in obtaining exposures
solely from food surveys, uncertainties in the nature of the
dose–response relationship, and uncertain- ties in HBV prevalence
data in different nations.
In this context, systematically analysing human stud- ies that
relate biomarkers of aflatoxin exposure and HBV infection to HCC
may provide a more precise and accurate measurement of burden of
HCC caused by aflatoxin. Therefore, in this study, we
systematically reviewed epidemiological studies on these
associations in different world regions. By combining the relevant
odds ratios (ORs) and relative risks (RRs) from these
Fig. 1. Selection of studies for in
studies, we conducted meta-analyses to calculate popu-
lation-attributable risk (PAR) of aflatoxin-related HCC in the
population overall, as well as in HBV+ and HBV populations. PAR is
the proportion of dis- ease cases that could be avoided if a
particular risk fac- tor was eliminated in a population. In the
context of our study, PAR of aflatoxin-related HCC is the
proportion of HCC cases that could be avoided in a chosen popula-
tion by reducing aflatoxin exposures (as measured by biomarkers)
from detectable to undetectable levels.
2. Methods
2.1. Search strategy
We performed a literature search until May 13th, 2011, using the
following search terms on Medline/Pub- Med: (aflatoxin) and
(hepatitis B) and (liver cancer); (aflatoxin) and (hepatitis B) and
(hepatocellular carci- noma). Additionally, we searched reference
lists from retrieved articles to identify further relevant studies.
Our systematic review and meta-analyses were con- ducted in
adherence to PRISMA standards for report- ing
meta-analyses.14
2.2. Eligibility criteria
Studies were included in the systematic review if they met the
following criteria: (1) case-control or cohort study design; (2)
aflatoxin as the exposure of interest; (3) HBV as the infection of
interest (hepatitis B virus surface antigen [HBsAg] as a marker of
chronic HBV infection); (4) HCC as the outcome of interest; and (5)
relative risk (RR) or odds ratio (OR) estimates with 95% confidence
intervals (CIs) reported, or data to cal- culate these.
clusion in systematic review.
No Source Location/
(cohort of 18,244
1986–1992
M 45–64 50 cases (36%) 267 matched controls (12%) AFB1-N7-Gua
adduct (detectable vs
non-detectable, 0.07 ng aflatoxins/ml
smoking
50 cases (72%) 267 matched controls (41%) Multiple urinary
biomarker
(detectable vs non-detectable,
township cohort nested
1991–1992
F/M 36–65 20 cases (65%) 86 matched controls (37%) AFB1-albumin
adducts (detectable Vs
non-detectable, 0.01fmol/lg)
history of liver cancer cirrhosis
3 Chen et al., 199619
(nested case-control in
exposure)
detectable, 0.01fmol/lg)
consumption
township cohort nested
1991–1995
F/M 30–64 52 cases (60%) 168 matched controls (37%) AFB1-albumin
adducts (detectable vs
non-detectable, 0.01fmol/lg)
1.6 (0.4–5.5) HBsAg positivity
38 cases (53%) 137 matched controls (45%) Urinary aflatoxin
metabolite (high vs
low, 0.01fmol/lg)
(Hospital-based case-
control study)
China, 1994–
controls (2%)
liver diseases, and peanut
controls (49%)
liver diseases, and corn
case-control of a cohort
of 4841 male HBsAg
1988–1994
M 30–65 42 cases (29%) 43 matched controls (14%) AFB1-N7-gua,
(below 0.21 ng/ml Vs
0.21–0.36 ng/ml, 0.05 ng aflatoxin/ml
urine)
habitual alcohol drinking and
cigarette smoking status
42 cases (14%) 43 matched controls (16%) AFB1-N7-gua (below 0.21
ng/ml
Vs > 0.36 ng/ml, 0.05 ng aflatoxin/ml
urine)
2.8 (0.6–12.9)
42 cases (24%) 43 matched controls (23%) AFM1 (below 1.61 ng/ml Vs
1.61–
2.85 ng/ml, 0.05 ng aflatoxin/ml
urine)
1.9(0.5–7.2)
42 cases (55%) 43 matched controls (35%) AFM1 (below 1.61
ng/ml
Vs > 2.85 ng/ml, 0.05 ng aflatoxin/ml
urine)
(case-control study)
1984–1995
F/M 105 cases (80%) 37 controls (43%) AFB1-DNA adducts Corrected
OR:
3.9(1.4–11.5)
(case-control study)
The Gambia,
1997–1998
F/M 20–73 53 cases (36%) 53 matched controls (5.7%) Ser-249 P53
mutation 16.4 (3.0–90.5) Age, sex, recruitment site and
HBsAg positivity
township cohort nested
1991–1997
F/M 30–64 75 cases (64%) 140 matched controls (46%)
Aflatoxin-albumin adducts
(detectable vs non-detectable,
10 Omer et al., 200124
(case-control study)
Sudan 1996–
1998
F/M 20–70 115 cases 199 matched controls Peanut butter
consumption
> 300 g/mo Vs Peanut butter
consumption < 70 g/mo
(continued on next page)
(Hospital-based cohort,
follow up
history of HCC
(case-control study)
Qidong,
China
F/M 19–87 25 cases (40%) 30 controls (6.7%) Ser 249 TP 53 mutation
22.1(3.2–91.7) Sex, age, recruitment
site and HBsAg
(case-control study)
Sudan, 1996–
(26%)
control study)
(3.4%)
group, season of
recruitment and daily
(hospital-based case-
Low (6 100 lmol/
mol DNA) Vs
controls
(17%)
AFB1-
adduct:
township cohort nested
F/M 30–64 yr 230 cases (93%) 1052 matched controls
(95%)
habitual smoking,
(33%)
(P598),
(88%)
(44%)
the mean
(68%)
a All the eligible studies were conducted in China,6 Taiwan,7 or
sub-Saharan Africa.4 Fourteen studies reported biomarker
measurements for aflatoxin exposure, while the other three studies
relied on food consumption data.
Twelve studies included both HBsAg+ and HBsAg individuals, with
risk estimates that were adjusted for HBsAg positivity (nine
studies). Five studies were conducted in HBsAg+ populations only. b
Amongst the fourteen studies that utilised biomarkers, five
measured urinary aflatoxin biomarkers, including AFM1 and
AFB1-N7-Guanine, six measured AFB1-albumin adducts, two measured
AFB1-DNA adducts, and
three measured TP53 249ser mutations. Several studies included
measures of more than one biomarker. c 15 out of 16 identified
case-control studies provided matched ORs.
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2129
2.3. Data extraction
The following data were extracted from each study: authors,
publication year, study design and sample size, study location,
study period, participants’ gender and age range, metric and range
of aflatoxin exposure, esti- mated adjusted RRs/ORs and variables
adjusted for analysis. Because all identified studies are
case-control designs except one cohort study, and because RR and OR
can be used interchangeably when the disease is rel- atively rare
(<15%; HCC rates are lower than this in the populations
studied), we combined the RR from this study with the ORs from the
case-control studies to cal- culate a summary OR. If aflatoxin
exposure was mea- sured using different biomarkers in the same
study, we selected the ones reflecting consistent biomarkers
amongst different studies (one OR per study was used).
2.4. Statistical methods for meta-analysis
The ORs from the studies were first combined in the meta-analysis
using a random-effects model, and then a fixed-effects model if
heterogeneity in the study pool was insignificant.15 The studies
were categorised by the recruited population type: general
populations, and HBV+ or HBV populations. First, all the studies
pro- viding data for general populations (including both HBV+ and
HBV individuals) were combined, and ORs of aflatoxin-related HCC
after HBsAg+ adjust- ment and ORs for combined (aflatoxin + HBV)
effects were analysed. Then the studies with data from HBV+
populations (and studies that recruited from the general population
but separately estimated ORs in HBV+ pop- ulations) were combined;
and the ORs for HBV+ pop- ulations only were estimated. We also
combined the studies that separately estimated the ORs in HBV pop-
ulations. If the study examined the association between aflatoxin
exposure and HCC in various exposure catego- ries, we chose the ORs
reflecting highest and lowest lev- els of aflatoxin exposure for
the meta-analysis.
Heterogeneity amongst the studies was evaluated using the Cochran’s
Q value calculated from the Man- tel–Haenszel method and the I2
statistic.15 We per- formed sensitivity analyses in which each
study was in turn removed and the rest analysed to evaluate if the
results were significantly affected by one particular study.
Publication bias was assessed by a funnel plot and associated
statistical tests of asymmetry. All statisti- cal analyses were
performed with Comprehensive Meta- Analysis software Version
2.2.
2.5. Statistical methods for PAR calculations
We estimated the PAR for aflatoxin-related HCC in HBV+ and HBV
populations for each study if the data were available. To estimate
the PAR for afla-
toxin-related HCC using the adjusted ORs, we used the attributable
fraction formula16:
AF POP ¼ Xz
1þ P iðRRi 1Þ
where AF POP is aflatoxin attributable risk fraction in the
population including exposed and unexposed individu- als, Pi is the
proportion of the population in stratum i
that is exposed, and Wi is the proportion of diseased individuals
(cases) in stratum i. We use adjusted ORi
in stratum i as an approximation of RRi. If the study provided risk
estimates adjusted by
HBsAg positivity, we used the formula below16 to esti- mate the PAR
of aflatoxin-related HCC in the general population:
AF POP ¼ P cðRR 1Þ
RR
where Pc is the proportion of cases exposed in the com- bined
population based on detection limits for aflatoxin biomarkers in
the studies, and HBsAg positivity-ad- justed OR is used as an
approximation of RR. For each AFpop, we calculated 95% confidence
intervals (CI) using the method described in Daly.17
3. Results
3.1. Literature search
The step-by-step process of our literature search is presented in
Fig. 1. From 479 results, we excluded human cell line studies,
animal studies, and review arti- cles. Using the eligibility
criteria described above, 27 studies were selected. Three more
relevant studies were identified from the reference lists of the 27
selected stud- ies. We then read the full texts of these 30
studies. Six studies were excluded because they were duplicated
reports from the same population in the same time per- iod, and
seven more were excluded because quantitative measurements of
association between aflatoxin exposure and HCC were not provided.
Thus, 17 studies were included in this systematic review and PAR
analysis.
3.2. Study characteristics
Table 1 provides an overview of the eligible studies. The 17
studies5–8,18–30 on aflatoxin exposure and HCC risk – eight
case-control studies, eight nested case-con- trol studies, and one
cohort study – were published between 1994 and 2009. There were
1680 HCC cases and 3052 controls in total.
Four studies reported results for one Taiwanese cohort from four
different time periods7,18,25,30 from 1980s to 2000s. To determine
if all these studies should be included in the meta-analysis, we
first examined the heterogeneity between the risk estimates
provided by these studies. Because of the significant
heterogeneity
Table 2 Summary of combined odds ratios in the meta-analysis.
Risk factor Study Population Study area (n of studies)
Cases/controlsa Odds Ratio, 95% CI Model Heterogeneity
Aflatoxin only General population with HBsAg+ adjustment
China4,6,22,27,28 634 cases/913 controls 5.99 (3.70–9.69) Fixed Q =
4.86, P = 0.18, I2 = 38.32 Taiwan3,7,18,30 b 198 cases/904 controls
2.01 (1.40–2.89) Fixed Q = 3.19, P = 0.20, I2 = 37.29 Sub-Saharan
Africa2,23,24 168 cases/252 controls 4.62 (2.12–10.08) Fixed Q =
269, P = 01, I2 = 6282 Summary9 1000 cases/2069 controls 4.75
(2.78–8.11) Random Q = 32.73, P < 0000, I2 = 75.56
General population with HBsAg+ adjustment after adjust
heterogeneity
Summary8,5–8,18,22,27,28 840 cases/1302 controls 5.72 (4.42–7.40)
Fixed Q = 8.40, P = 0.30, I2 = 16.66
General population with HBsAg+ adjustment by only including Wu et
al. as follow-up for cohort in Taiwan
Summary7,5,6,8,22,27,28,30 1000 cases/2069 controls 4.88
(2.62–9.10) Random Q = 32.55, P < 0.000, I2 = 81.57
General population with HBsAg+ adjustment by taking the average
effect of the series of Taiwan Studiesc
Summary7 1000 cases/2069 controls 4.92 (2.74–8.82) Random Q =
29.48, P < 0.000, I2 = 79.65
HBsAg+ individuals China3,6,26,29 189 cases/268 controls 2.00
(0.84–4.75) Random Q = 1066, P = 0005, I2 = 8124
Taiwan6,7,19–21,25,30 254 cases/310 controls 1.81(1.29–2.56) Fixed
Q = 8.38, P = 0.14, I2 = 40.35 Sub-Saharan Africa2,5,8 128 cases/56
controls 6.48 (0.22–194) Random Q = 6.54, P = 0.01, I2 = 84.71
Summary11d 571 cases/634 controls 2.39 (1.50–3.82) Random Q =
27.99, P = 0.002, I2 = 64.27
HBsAg+ individuals after adjust heterogeneity
Summary9,5–8,19–21,25,26 377 cases/383 controls 2.90 (2.09–4.01)
Fixed Q = 11.16, P = 0.19, I2 = 28.29 HBsAg+ individuals by only
including most recent follow-up studies in a cohort of Taiwan
Summary8,5,6,8,20,21,26,29,30 571 cases/634 controls 2.27
(1.24–4.14) Random Q = 24.33, P = 0.001, I2 = 71.23
HBsAg+ individuals by only combing studies with adjusted ORs
Summary 332 cases/538 controls 2.10 (1.25–3.52) Random Q = 16.40, P
= 0.012, I2 = 63.42
HBsAg+ individuals by taking the average effect of all follow-up
studies in the same cohorte
Summary8 571 cases/634 controls 2.35(1.38–3.99) Random Q = 2317, P
= 0002, I2 = 69.79
HBsAg individuals China1,6 18 cases/ 236 controls 34 (113–1025) / /
Taiwan3,7,20,30 81 cases/664 controls 500 (222–1128) Fixed Q = 369,
P = 016, I2 = 4579 Sub-Saharan Africa2,5,8 122 cases/391 controls
840 (415–1699) Fixed Q = 840, P = 019, I2 = 4263 Summary6 221
cases/1291 controls 591 (366–955) Fixed Q = 751, P = 019, I2 =
3342
HBsAg- individuals excluding Wu et al.30 Summary5 172 cases/769
controls 637 (374–1086) Fixed Q = 711, P = 013, I2 = 4371 HBV only
General population Summary6,5–8,20,30 244 cases/1072 controls 112
(748–167) Fixed Q = 237, P = 080, I2 = 000
General population after adjusted heterogeneity Summary5,5–8,20 171
cases/638 controls 113 (675–189) Fixed Q = 236, P = 067, I2 = 000
Aflatoxin and
HBV infection combined effects
General population Summary6,5–8,20,30 554 cases/1456 controls 541
(213–1377) Random Q = 1365, P = 002, I2 = 6336 General population
after adjust heterogeneity Summary5,5–8,20 452 cases/847 controls
730 (360–1483) Fixed Q = 348, P = 048, I2 = 000
a If there was a series of follow-up studies in the same cohort
need to be combined, only the numbers of cases and controls from
the largest follow-up study were counted, although different odds
ratios from different follow-up studies were combined to assess the
effect. All the cases and controls were only counted once, and as
well as in calculations presented in Tables 4 and 5.
b This row shows the summary odds ratio of combing three follow-up
studies in a Taiwan cohort in different years. c The summary odds
ratio obtained for the Taiwan cohort was used to represent the
effect of all studies in this cohorts, and combine with other
studies. d Seven studies7,15,17,21,22,25,26 reported adjusted ORs
on aflatoxin-related HCC risk in HBsAg+ individuals. Four
studies5,6,8,16 (including two studies conducted in Sub-Saharan
Africa countries) did
not provide adjusted ORs directly, but provided data to calculate
the unadjusted ORs. We calculated the unadjusted ORs for each of
these studies and combined them with ORs from other studies with
eligible data, thus we can include the effects of studies in
Sub-Saharan Africa population. In subgroup analysis, the large
variation of summarised ORs of aflatoxin-related HCC in HBsAg+
individuals may be explained by combining the unadjusted ORs. The
heterogeneity was significant when studies were combined to examine
the association between aflatoxin exposure and HCC risk in the
general population and in HBsAg+ individuals.
e The summary odds ratio obtained from different follow-up studies
for the Taiwan cohort was used to represent the effect of all
studies in this cohorts, and combine with other studies.
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Study name Statistics for each study Odds ratio and 95% CI
Odds Lower Upper Relative ratio limit limit Z-Value p-Value
weight
8.86
10.58
23.35
27.23
29.98
Favours A Favours B
Fixed Effect Model
Study name Statistics for each study Odds ratio and 95% CI
Relative Odds Lower Upper ratio limit limit Z-Value p-Value
weight
6.13
7.82
8.13
18.33
59.59
Favours A Favours B
Fixed Effect Model
Study name Statistics for each study Odds ratio and 95% CI
Relative Odds Lower Upper ratio limit limit Z-Value p-Value
weight
11.33
11.47
17.16
29.08
30.95
6.37 3.74 10.86 6.81 0.00
Omer et al., 2005 32.20 4.02 258.10 3.27 0.00
Wang et al., 1996 22.80 3.61 143.90 3.33 0.00
Lunn et al., 1997 17.00 2.79 103.56 3.07 0.00
Qian et al., 1994 7.30 2.19 24.31 3.24 0.00
Kirk et al., 2005 10.00 5.13 19.49 6.76 0.00
11.29 6.75 18.90 9.22 0.00
Kirk et al.,2005 399.00 48.64 3272.87 5.58 0.00
Wang et al., 1996 111.90 13.82 906.18 4.42 0.00
Lunn et al., 1997 67.60 12.22 373.88 4.83 0.00
Omer et al., 2004 41.50 11.16 154.38 5.56 0.00
Qian et al., 1994 59.40 16.62 212.28 6.29 0.00
73.02 35.95 148.30 11.87 0.00
0.01 0.1 1 10 100
Favours A Favours B
Fixed Effect Model
2C. Odds Ratios (for individual study and pooled studies) of Liver
Cancer from Combined Effects Excluding Wu et al.
2B. Odds Ratios (for individual study and pooled studies) of Liver
Cancer from HBV+ Excluding Wu et al.
2A. Odds Ratios (for individual study and pooled studies) of Liver
Cancer from Aflatoxin Exposure Excluding Wu et al.
Fig. 2. Odds ratios (ORs) and 95% CIs for association between liver
cancer and two risk factors (aflatoxin exposure and chronic HBV),
independently and in combination. Squares and horizontal lines
correspond to the study-specific OR and 95% CI; the box size is
proportional to the meta-analysis study weight; diamonds represent
summarised ORs. 2A: ORs with 95% CI for association between liver
cancer and chronic HBV+ only, excluding Wu et al.30 2B: ORs with
95% CI for association between liver cancer and aflatoxin exposure
only, excluding Wu et al.30 2C: ORs with 95% CI for association
between liver cancer and the combination effects of two risk
factors, excluding Wu et al.30
Y. Liu et al. / European Journal of Cancer 48 (2012) 2125–2136
2131
of aflatoxin exposures and HCC risk estimates in this cohort
between the follow-up studies through the years, we treated these
as independent studies in the analysis. In analyses that included
only the most recent of all studies
in a particular cohort, the results were nearly identical to those
obtained when including all studies (Table 2). Two articles
reported results from one case-control study in Sudan from
different perspectives (risk estimates for
0.0
0.2
0.4
Funnel Plot of Standard Error by Log odds ratio
2132 Y. Liu et al. / European Journal of Cancer 48 (2012)
2125–2136
the general population after adjustment of HBsAg+, and risk
estimates for HBsAg+ or HBsAg separately).8,24
Likewise, two articles reported results from a study in the Gambia
with risk estimates for the general popula- tion after adjustment
of HBsAg+, and risk estimates for HBsAg+ or HBsAg
separately.5,23
-4 -3 -2 -1 0 1 2 3 4
0.6
0.8
1.0
Log odds ratio
Fig. 3. Funnel plot to assess possible publication or other
selection bias for the association between aflatoxin exposure and
liver cancer risk in general population. No statistically
significant asymmetry was found. Each circle represents 1 study. 10
studies6,7,18,20,22–24,27,28,30 are eligible for this plot. 7
studies not included (5 only studied the association in HBsAg+
individuals, and 2 are duplicate studies included in meta-analysis
for different data extraction purpose, as explained in the Methods
section).
3.3. Aflatoxin exposure and HCC risk by HBsAg Status
The association between aflatoxin exposure and HCC, independently
or in conjunction with HBV, was analysed by combining eligible
studies by HBsAg+ sta- tus and calculating summary ORs (Table 2).
Meta-anal- yses were conducted by geographic region (China, Taiwan,
and sub-Saharan Africa).
Aflatoxin exposure is significantly associated with HCC risk,
regardless of HBsAg status, with a summa- rised OR of 4.75
(2.78–8.11) from nine studies in the general population adjusted by
HBsAg positivity, 2.39 (1.50–3.82) from eleven studies in HBsAg+
populations and 5.91 (3.66–9.55) from six studies in HBsAg
populations.
3.4. Sensitivity analysis
For the meta-analysis of aflatoxin-related HCC risk in the general
population, our sensitivity analyses revealed that Wu et al.30 was
the most influential study in determining the summarised OR. After
excluding this particular study, heterogeneity was significantly
reduced (Q = 8.40, P = 0.30, I2 = 16.66), and the summarised OR was
5.57 (3.78–7.79).
For the meta-analysis of aflatoxin exposure and HCC in HBsAg+
populations, our sensitivity analyses showed that two studies,
Szymanska et al.29 and Wu et al.,30 sub- stantially influenced the
summarised OR. After exclud- ing the two studies, heterogeneity was
significantly reduced (Q = 11.16, P = 0.19, I2 = 28.29), and the
sum- marised OR of HCC risk for detectable vs. non-detect- able
aflatoxin exposure in HBsAg+ individuals was 2.90 (2.09–4.01).
These results suggest that the two stud- ies that measured the
association between HCC and afla- toxin exposure in the most recent
years29,30 appear to have significantly different results from
relatively earlier studies.
For the 10 studies6,7,18,20,22–24,27,28,30 associating afla- toxin
and liver cancer in the general population, we assessed publication
or other forms of selection bias by a funnel plot (Fig. 2) and
associated statistical tests of funnel plot asymmetry.31 Seven
studies are not included in this plot; five studied the association
in HBsAg+ indi- viduals only, and two are duplicate studies
included in meta-analysis for different data extraction purposes,
as explained in the methods. The funnel plot provides little
evidence of an important departure from symmetry, indi- cating that
publication or other forms of selection bias
were not a serious limitation in our meta-analysis. This visual
impression of symmetry was corroborated by the statistical tests of
funnel plot asymmetry.
3.5. Multiplicative model of effects between aflatoxin
exposure and chronic HBV infection
The meta-analysis allowed us to quantitatively evalu- ate the model
of effects between the two risk factors afla- toxin and HBV in
liver cancer. The summary OR of six studies5–8,20,30 reporting ORs
of HCC risk from both aflatoxin exposure and HBV is 54.1
(21.3–137.7) with significant heterogeneity (Q = 13.65, P = 0.02,
I2 = 63.36). The summary OR of the same group of studies for HCC
from aflatoxin exposure alone is 5.91 (3.66–9.55), while the
summary OR on HCC risk from chronic HBV alone is 11.2 (7.48–16.7),
both with no sig- nificant heterogeneity. When we excluded Wu et
al.30
which contributes most to the heterogeneity, the sum- marised OR
for combined effects increased to 73.0 (36.0–148.3), 6.37
(3.74–10.86) for aflatoxin exposure alone, and 11.3 (6.75–18.9) for
chronic HBV infection alone (Fig. 3). These estimates indicate an
almost per- fectly multiplicative model of effects between
aflatoxin exposure and chronic HBV in HCC risk.
3.6. PAR of HCC from aflatoxin exposure in each study
population
The PAR of aflatoxin-related HCC was calculated for each study
population (Table 3). PAR is the proportion of the HCC cases that
could be prevented by reducing aflatoxin exposures to “control”
levels in each study. For example, HCC in the Chen et al.18
Taiwanese study population could be reduced by about 10% (2.5–12%)
if dietary aflatoxin exposures in this population were
Table 3 Population attributable risk of liver cancer caused by
aflatoxin exposure in HBV+ populations, HBV populations, and the
general population.
Studies Exposure measurement PAR for aflatoxin attributable HCC
risk in HBsAg+
PAR for aflatoxin attributable HCC risk in HBsAg
PAR for aflatoxin attributable HCC risk in general study population
adjusted by HBsAg+
Qian et al., 19946 (Shanghai, China)
Multiple urinary aflatoxin metabolites
40% (24–47%)a 3.6% (0.3–5.6%) 9.0% (5.9–10.4%)
Chen et al., 199618 (Taiwan) AFB1 albumin adducts n/a n/a 10%
(2.5–12%) Chen et al., 199619 (Taiwan) AFB1 albumin
adducts6Low
vs undetectable 4.2% (0–13%) n/a
HBV individuals only n/a HBV individuals only
AFB1 albumin adducts6High vs undetectable
4.5% (0–11%) n/a HBV individuals only
n/a HBV individuals only
n/a HBV individuals only
n/a HBV individuals only
Wang et al., 19967 (Taiwan) AFB1 albumin adducts 31% (0–51%) 0
(0–2.3%) 5% (0–11%) Urinary aflatoxin metabolites
41% (8.1–54%) 1% (0–4.1%) 11% (1.4% - 13.7%)
Lunn et al., 199720 (Taiwan) AFB1-DNA adduct 31% (0–75%)b 44%
(29%-47%) n/a Yu et al., 199721 (Taiwan) 1.61–285 ng/ml AFM1
vs
non-detectable) 2.1% (0–7.2%) n/a
HBV individuals only n/a HBV individuals only
>285 ng/ml AFM1 vs non- detectable)
19% (2.2–25%) n/a HBV individuals only
n/a HBV individuals only
Sum = 21% (2.2– 32%)
n/a HBV individuals only
n/a HBV individuals only
Zhang et al., 199722 (Henan, China)
Corn consumption n/a n/a 175% (8–18.4%) Peanut consumption n/a n/a
36% (16–45%)
Kirk et al., 200023 (The Gambia)
Ser 249 TP53 mutation n/a n/a 17% (12–18%)
Omer et al., 200124 (Sudan) Average peanut butter consumption
n/a n/a 23% (11–29%)
Sun et al., 2001 (Taiwan)25 AFB1 albumin adducts 12% (1.7% - 20%)
n/a HBV individuals only
n/a HBV individuals only
AFM1 57% (16–72%)c n/a n/a
Huang et al. 200327 (Qidong, China)
Ser 249 TP53 mutation n/a n/a 17% (13–18%)
Omer et al., 20048(Sudan) Average peanut butter consumption
5.4% (0–62%)d 20% (9.0–25%) n/a
Kirk et al., 20055 (The Gambia)
Ser 249 TP53 mutation 63% (39–67%)e 12% (6.3–17%) 13%
(12–14%)6f
Wu et al., 200930 (Taiwan) AFB1 albumin adducts 3.7% (0–11%) 1.7%
(0–4.6%) 2.1% (0.06–3.4%) urinary aflatoxin metabolites 3.1%
(0–11.7%) 4.7% (1.2–6.7%) 4.4% (1.6–6.3%)
Szymanska et al., 200929
HBV individuals only n/a HBV individuals only
Long et al., 200928 (Guangxi, China)
AFB1-DNA adduct medium vs low
n/a n/a 6.8% (4.5–8.5%)
AFB1-DNA adduct high vs low
n/a n/a 19% (17–20%)
Total n/a n/a 26% (22–29%)
a Calculated from unadjusted OR. b Calculated from unadjusted OR. c
Author estimated. d Calculated from unadjusted OR. e Calculated
from unadjusted OR. f Calculated from ORs unadjusted by
HBsAg+).
Y. Liu et al. / European Journal of Cancer 48 (2012) 2125–2136
2133
reduced such that aflatoxin-albumin adduct levels were below 0.01
fmol/lg (detection limit in this study), or if dietary aflatoxin
exposures could be decreased to below 4.3 ng/kg bw/day (biomarker
detection limit extrapo- lated to dietary exposure). HCC in the
study population of Shanghai males in Qian et al.6 could be reduced
by about 9.0% (5.9–10.4%) if aflatoxin exposures in this population
were reduced to below 6 ng/kg bw/day: the
average aflatoxin exposure level in the control group. Our results
showed that the PAR of HCC caused by aflatoxin is higher in HBV+
populations than in HBV populations.
In HBV+ populations in a Taiwanese cohort, the PAR for
aflatoxin-related HCC is consistently decreas- ing, as indicated by
a series of follow-up studies: 31% in 1980s7, 12% in 1990s,25 and
3% in 2000s.30 Overall,
Table 4 Estimated population attributable HCC risk from aflatoxin
exposure in the general population by combining the eligible
studies.
Study population Total exposed cases (n1)
Total sample size (n2)
PAR (95% CI)
General population adjusted by HBV status (6, 7, 18, 22–24, 27, 28,
30)
China 475 1588 0.299 5.99 (3.70–9.69) 25% (22–27%) Taiwan 113 1102
0.103 2.01 (1.40–2.89) 5.2% (2.9–6.7%) Sub-Saharan Africa 82 340
0.241 4.62 (2.12–10.08) 19% (13–22%) Summary 670 3030 0.221 475
(278–8.11) 17% (14–19%)
General population adjusted by HBV status after excluding Wu et al.
200930
Summary 583 2103 0.277 5.72(4.42–7.40) 23% (21–24%)
Table 5 Estimated population attributable HCC risk from aflatoxin
exposure in HBV+ and HBV populations by combining the eligible
studies.
Study population Total HBsAg+(or HBsAg) (n1)
Total HCC cases in HBsAg+(or HBsAg) (n2)
Total exposed HBsAg+(or HBsAg) (n3)
Proportion of HCC cases in HBsAg+(or HBsAg) (W1)
Proportion of exposed HBsAg+(or HBsAg) (P1)
Summarised OR (95%CI)
HBV+ population (5–8, 19– 21, 25, 26, 29, 30)
China 457 189 276 0.414 0.604 2.00(0.84– 4.75)
16% (0–29%)
14% (6.3–21%)
Sub- Saharan Africa
48% (0–69%)
21% (10–29%)
and Wu et al(30)
16% (9–20%)
24% (14–33%)
25% (18–30%)
Taiwan 745 81 332 0.109 0.446 5.00 (2.22– 11.28)
7% (3.8–89%)
15% (9.7–19%)
8.8% (6.7– 10%)
a Studies (including two studies in Sub-Saharan Africa countries)
with unadjusted ORs were also combined to calculate the overall
PAR, thus the Sub-Saharan study population can be included.
2134 Y. Liu et al. / European Journal of Cancer 48 (2012)
2125–2136
the PAR of aflatoxin-related HCC is decreasing in Tai- wan in both
HBV+ and HBV individuals, from as high as 44% in 1990s20 to 2% in
2000s.30
We combined all aflatoxin-exposed cases, HBV+ and HBV individuals,
and controls from all the eligible studies to calculate the PAR of
aflatoxin-related HCC by HBsAg status and world region (Tables 4
and 5). The PAR of aflatoxin-related HCC in the general popu-
lation after HBV adjustment is 17% (14–19%). Because the earlier
sensitivity analysis demonstrated that the remaining studies after
exclusion of Wu et al.30 do not have statistically significant
heterogeneity, we also calcu- lated the PAR of aflatoxin-related
HCC after exclusion of.30 The PAR increased to 23% (21–24%).
The PAR of aflatoxin-related HCC in the HBV+ population is 21%
(10–29%). A separate calculation was performed excluding Szymanska
et al.29 and Wu et al.,30 the most influential studies indicated by
the sen- sitivity analysis. The new PAR of aflatoxin-related HCC in
the HBV+ population was 25% (18–30%). The PAR of aflatoxin-related
HCC in HBV populations is 8.8% (6.7–10%).
4. Discussion
Aflatoxin exposure is significantly associated with HCC risk
regardless of HBV status. Our meta-analyses show that in areas of
high aflatoxin exposure and
Y. Liu et al. / European Journal of Cancer 48 (2012) 2125–2136
2135
chronic HBV infection, aflatoxin exposure and HBV have a nearly
perfectly multiplicative relationship in increasing HCC risk. In
populations including both HBV+ and HBV individuals in the
geographic regions studied, the PAR of aflatoxin-related HCC was
esti- mated at 17% (14–19%). This implies that if it were pos-
sible to reduce aflatoxin to below detectable limits in these
regions, HCC incidence could be reduced by 14– 19%. There are
roughly 520,000 new HCC cases in China, southeastern Asia and
sub-Saharan Africa each year.32 If the PARs are generalised to
these areas, the implication is that, by reducing aflatoxin in
human diets to below detectable levels, 72,800 to 98,800 new HCC
cases could be prevented every year. If this PAR was generalised to
regions of the world beyond Africa and Asia, the overall number of
HCC cases (749,000 new cases per year32) that could be prevented by
aflatoxin control would reach 105,000–142,000.
The PAR of aflatoxin-related HCC increases to 23% (21–24%), and
heterogeneity amongst the studies decreases significantly, if one
study30 is excluded from the meta-analysis. However, this study is
important because it suggests that aflatoxin exposure is decreasing
over time in the Taiwanese (Penghu) population studied. Our PAR
estimates for individual studies showed a decrease in PAR of
aflatoxin-related HCC in the Penghu cohort in the last three
decades. It is worth noting that in a 1970s food survey, over
one-third of peanuts in Pen- ghu were heavily contaminated by
aflatoxins, with an average aflatoxin content of 167 lg/kg.33 Mean
urinary aflatoxin in HCC patients in this cohort form was 219 lg/ml
in 1991/1992,7,18 and decreased to 0.017 lg/ ml in HCC patients in
the same cohort in 2004.30 Also, the HBV vaccination programme in
Taiwan has success- fully reduced HBV prevalence, further reducing
HCC risk.34
In some parts of the world such as Taiwan, aflatoxin exposure is
decreasing. In other parts of the world such as Africa, rural
China, and Southeast Asia, there is little evidence that aflatoxin
exposure is decreasing; in fact, two recent Kenyan events of
extremely high aflatoxin levels in maize (in 2004–2005, and again
in 2010) suggest the opposite. With climate change, aflatoxin
contamina- tion in food crops may become exacerbated due to the
conditions favoring proliferation of Aspergilli.35 Hence, further
efforts to reduce aflatoxin-related disease are needed in high-risk
areas of the world.
There are several limitations in this analysis. First, the
epidemiological studies included were conducted in areas of the
world with both high aflatoxin and HBV (Asia and sub-Saharan
Africa). Thus, although these regions account for most of the
aflatoxin-induced HCC cases worldwide,13 the estimated PAR is not
nec- essarily applicable in areas with much lower aflatoxin
exposures. Second, odds ratios from studies employing food surveys,
exposure biomarkers and biological effect
biomarkers were combined. This decreases the precision of the
analysis, as different biomarkers have different detection limits
and measure different endpoints, and food surveys are less precise
than biomarkers for expo- sure estimation. Third, the PAR is meant
to represent the proportion by which the disease could be reduced
if the risk factor in question was removed. It is not pos- sible to
instantaneously reduce aflatoxin to below detectable limits
worldwide – rather, the PAR calculated is meant to estimate the
burden of HCC caused by one risk factor (aflatoxin) and to project
the extent to which the problem could be reduced in future
generations if aflatoxin control strategies were widespread.
In summary, this study is the first to quantitatively evaluate the
model of effects between aflatoxin and HBV in inducing liver cancer
by combining results from multiple epidemiological studies. The
range of PARs calculated in this analysis, 14–19% (21–24% excluding
one study contributing to heterogeneity), is consistent with our
previous report of 5–28% using a different methodology
(quantitative cancer risk assessment).13
The PAR of aflatoxin-related HCC is higher in HBsAg + populations
than HBsAg- populations. In recent years, the PAR of
aflatoxin-related HCC has shown a decreasing trend in areas such as
Taiwan, indi- cating the benefits of reduced aflatoxin exposure and
HBV prevalence by public health interventions.
Conflict of interest statement
The authors declare that they have no competing financial
interests.
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Population attributable risk of aflatoxin-related liver cancer:
Systematic review and meta-analysis
1 Introduction
2 Methods
2.5 Statistical methods for PAR calculations
3 Results
3.4 Sensitivity analysis
3.5 Multiplicative model of effects between aflatoxin exposure and
chronic HBV infection
3.6 PAR of HCC from aflatoxin exposure in each study
population
4 Discussion